Background: Basolateral K+ channels hyperpolarize colonocytes to ensure Na+ (and thus water) absorption. Small conductance basolateral (KCNQ1/KCNE3) K+ channels have never been evaluated in human colon. We therefore evaluated KCNQ1/KCNE3 channels in distal colonic crypts obtained from normal and active ulcerative colitis (UC) patients.Methods: KCNQ1 and KCNE3 mRNA levels were determined by qPCR, and KCNQ1/KCNE3 channel activity in normal and UC crypts, and the effects of forskolin (activator of adenylate cyclase) and UC-related proinflammatory cytokines on normal crypts, studied by patch clamp recording.Results: Whereas KCNQ1 and KCNE3 mRNA expression was similar in normal and UC crypts, single 6.8 pS channels were seen in 36% of basolateral patches in normal crypts, and to an even greater extent (74% of patches, P < 0.001) in UC crypts, with two or more channels per patch. Channel activity was 10-fold higher (P < 0.001) in UC crypts, with a greater contribution to basolateral conductance (5.85 +/- 0.62 mS cm(-2)) than in controls (0.28 +/- 0.04 mS cm(-2), P < 0.001). In control crypts, forskolin and thromboxane A(2) stimulated channel activity 30-fold and 10-fold respectively, while PGE(2), IL-1 beta, and LTD4 had no effect.Conclusions: KCNQ1/KCNE3 channels make only a small contribution to basolateral conductance in normal colonic crypts, with increased channel activity in UC appearing insufficient to prevent colonic cell depolarization in this disease. This supports the proposal that defective Na+ absorption rather than enhanced Cl- secretion, is the dominant pathophysiological mechanism of diarrhea in UC. (C) 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.orgilicensesiby/4.0/).

Introduction and Aims: Basolateral K+ channels keep colonic cells in a hyperpolarized state to ensure transcellular ionic movements. Intermediate conductance K+ (IK) channels dominate the basolateral membrane conductance in human colonic cells, but their expression and activity are markedly decreased in ulcerative colitis (UC), thus contributing to defective Na+ and water absorption across inflamed mucosa (J Pathol 2012;226:463-470). This study is the first to characterize basolateral small conductance (KCNQ1/KCNE3) K+ channels in normal human colon, and explore their activity in UC. Methods: Intact crypts were isolated from distal colonic biopsies taken while investigating patients with functional diarrhea (normals) and patients with UC not receiving steroids. Expression of KCNQ1 and KCNE3 mRNAs was determined by qPCR. Patch clamp recording was used to study (i) basolateral KCNQ1/KCNE3 channel activity in normal and UC crypt cells and (ii) in normal crypts cells, the effects of forskolin (an activator of adenylate cyclase) and inflammatory mediators present at high concentrations in UC. Results: qPCR product bands were of the expected size (KCNQ1 160 bp, KCNE3 119 bp, β-actin 148 bp). Product identity was confirmed by automated sequencing and melting curve analysis, and KCNQ1 and KCNE3 mRNA expression was similar in normal and UC patients. In normal crypts (n=34), usually single channels (conductance 6.8 ± 0.5 pS) were seen in 36% of patches, whereas in UC crypts (n=9), two or more channels were seen in 74% of patches, the difference in channel prevalence between the two groups being significant (P<0.001). Overall channel activity was 10-fold greater in UC than control patients (P<0.001). These channels constituted a greater calculated basolateral conductance (GSK) in UC (5.85 ± 0.62 mS.cm-2) than in control patients (0.28 ± 0.04 mS.cm-2, P<0.001), but this increase in GSK did not compensate for the decrease in basolateral membrane conductance (from 22 mS.cm-2 to 5 mS.cm-2) previously attributed to decreased IK channel expression/activity in UC (J Pathol 2012;226:463-470). In control crypts, 10μM forskolin and 1μM thromboxane A2 stimulated channel activity 30-fold (n=8, P<0.005) and 10-fold (n=5, P<0.05) respectively, while 100nM PGE2, 10nM IL-1β and 1μM LTD4 had no effect. Conclusions: Crypt cells in normal human colon express cAMP-activated KCNQ1/ KCNE3 channels which make a far smaller contribution to overall basolateral conductance than IK channels. In active UC, KCNQ1/KCNE3 channel activity is significantly increased, but this fails to compensate for the depolarizing effect of decreased IK channel expression/ activity. This may explain, at least in part, why defective electrogenic Na+ absorption rather than increased electrogenic Cl secretion is the dominant pathophysiological mechanism of diarrhea in active UC (J Pathol 2012;226:463-470).

Background Human colon may secrete substantial amounts of water secondary to chloride (Cl−) and/or potassium (K+) secretion in a variety of diarrhoeal diseases. Ion secretion occurs via Cl− and K+ channels, which are generally assumed to be co-located in the colonocyte apical membrane, although their exact cellular sites remain unclear. Objective To investigate the location of apical Cl− (CFTR) and apical K+ (large conductance; BK) channels within human colonic epithelium. Design Whole-cell patch clamp recordings were obtained from intact human colonic crypts. Specific blockers of K+ channels and CFTR identified different types of K+ channel and CFTR under resting conditions and after stimulating intracellular cAMP with forskolin. The BK channel β3-subunit was localised by immunostaining. Results Two types of crypt cells were identified. One (73% of cells) had whole-cell currents dominated by intermediate conductance (IK) K+ channels under resting conditions, which developed large CFTR-mediated currents in response to increasing intracellular cAMP. The other (27% of cells) had resting currents dominated by BK channels inhibited by the BK channel blocker penitrem A, but insensitive to both forskolin and the IK channel blocker clotrimazole. Immunostaining showed co-localisation of the BK channel β3-subunit and the goblet cell marker, MUC2. Conclusions In human colon, Cl− secretion originates from the dominant population of colonocytes expressing apical CFTR, whereas K+ secretion is derived from a smaller population of goblet cells expressing apical BK channels. These findings provide new insights into the pathophysiology of secretory diarrhoea and should be taken into account during the development of anti-diarrhoeal drugs.

Diarrhoea in ulcerative colitis (UC) mainly reflects impaired colonic Na+ and water absorption. Colonocyte membrane potential, an important determinant of electrogenic Na+ absorption, is reduced in UC. Colonocyte potential is principally determined by basolateral IK (KCa3.1) channel activity. To determine whether reduced Na+ absorption in UC might be associated with decreased IK channel expression and activity, we used molecular and patch clamp recording techniques to evaluate IK channels in colon from control patients and patients with active UC. In control patients, immunolabelling revealed basolateral IK channels distributed uniformly along the surface‐crypt axis, with substantially decreased immunolabelling in patients with active UC, although IK mRNA levels measured by quantitative PCR were similar in both groups. Patch clamp analysis indicated that cell conductance was dominated by basolateral IK channels in control patients, but channel abundance and overall activity were reduced by 53% (p = 0.03) and 61% (p = 0.04), respectively, in patients with active UC. These changes resulted in a 75% (p = 0.003) decrease in the estimated basolateral membrane K+ conductance in UC patients compared with controls. Levels of IK channel immunolabelling and activity in UC patients in clinical remission were similar to those in control patients. We conclude that a substantial decrease in basolateral IK channel expression and activity in active UC most likely explains the epithelial cell depolarization observed in this disease, and decreases the electrical driving force for electrogenic Na+ transport, thereby impairing Na+ absorption (and as a consequence, Cl− and water absorption) across the inflamed mucosa. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Major liver resection is associated with impaired intestinal perfusion and intestinal ischemia, resulting in decreased mucosal integrity, increased bacterial translocation, and an increased risk of postoperative sepsis. However, the mechanism by which ischemia impairs intestinal mucosal integrity is unclear. We therefore evaluated the role of Ca2+-sensitive, intermediate-conductance (IKCa) basolateral potassium channels in enhanced intestinal permeability secondary to chemical hypoxia. The effects of chemical hypoxia induced by 100 μM dinitrophenol (DNP) and 5 mM deoxyglucose (DG) on basolateral IKCa channel activity and whole cell conductance in intact human colonic crypts, and paracellular permeability (GS) in isolated colonic sheets, were determined by patch-clamp recording and transepithelial electrical measurements, respectively. DNP and DG rapidly stimulated IKCa channels in cell-attached basolateral membrane patches and elicited a twofold increase ( P = 0.004) in whole cell conductance in amphotericin B-permeabilized membrane patches, changes that were inhibited by the specific IKCa channel blockers TRAM-34 (100 nM) and clotrimazole (CLT; 10 μM). In colonic sheets apically permeabilized with nystatin, DNP elicited a twofold increase ( P = 0.005) in GS, which was largely inhibited by the serosal addition of 50 μM CLT. We conclude that, in intestinal epithelia, chemical hypoxia increases GS through a mechanism involving basolateral IKCa channel activation. Basolateral IKCa channel inhibition may prevent or limit increased intestinal permeability during liver surgery.

The human colon has the capacity to secrete potassium (K(+)) ions and enhanced K(+) secretion is a feature of a variety of diarrhoeal diseases. Recent work points to K(+) secretion in human colon being mediated by high conductance (BK) K(+) channels located in the apical membrane of colonic epithelial cells. The aim of this review is to highlight the importance of these channels in maintaining K(+) homoeostasis in health and disease.

Decreased sodium (Na+), chloride (Cl−), and water absorption, and increased potassium (K+) secretion, contribute to the pathogenesis of diarrhoea in ulcerative colitis. The cellular abnormalities underlying decreased Na+ and Cl− absorption are becoming clearer, but the mechanism of increased K+ secretion is unknown. Human colon is normally a K+ secretory epithelium, making it likely that K+ channels are expressed in the luminal (apical) membrane. Based on the assumption that these K+ channels resembled the high conductance luminal K+ (BK) channels previously identified in rat colon, we used molecular and patch clamp recording techniques to evaluate BK channel expression in normal and inflamed human colon, and the distribution and characteristics of these channels in normal colon. In normal colon, BK channel α‐subunit protein was immunolocalized to surface cells and upper crypt cells. By contrast, in ulcerative colitis, although BK channel α‐subunit protein expression was unchanged in surface cells, it extended along the entire crypt irrespective of whether the disease was active or quiescent. BK channel α‐subunit protein and mRNA expression (evaluated by western blotting and real‐time PCR, respectively) were similar in the normal ascending and sigmoid colon. Of the four possible β‐subunits (β1–4), the β1‐ and β3‐subunits were dominant. Voltage‐dependent, barium‐inhibitable, luminal K+ channels with a unitary conductance of 214 pS were identified at low abundance in the luminal membrane of surface cells around the openings of sigmoid colonic crypts. We conclude that increased faecal K+ losses in ulcerative colitis, and possibly other diseases associated with altered colonic K+ transport, may reflect wider expression of luminal BK channels along the crypt axis. Copyright © 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

ATP-sensitive potassium (KATP) channels play a central role in glucose-stimulated insulin secretion (GSIS) by pancreatic beta-cells. Activity of these channels is determined by their open probability (Po) and the number of channels present in a cell. Glucose is known to reduce Po, but whether it also affects the channel density is unknown. Using INS-1 model beta-cell line, we show that the expression of K(ATP) channel subunits, Kir6.2 and SUR1, is high at low glucose, but declines sharply when the ambient glucose concentration exceeds 5mM. In response to glucose deprivation, channel synthesis increases rapidly by up-regulating translation of existing mRNAs. The effects of glucose deprivation could be mimicked by pharmacological activation of 5'-AMP-activated protein kinase with 5-aminoimidazole-4-carboxamide ribonucleotide and metformin. Pancreatic beta-cells which have lost their ability for GSIS do not show such changes implicating a possible (patho-)physiological link between glucose-regulated KATP channel expression and the capacity for normal GSIS.

Aldosterone produces rapid, non-genomic, inhibition of basolateral intermediate conductance K(+) (IK(Ca)) channels in human colonic crypt cells but the intracellular second messengers involved are unclear. We therefore evaluated the role of protein kinase C (PKC) in aldosterone's non-genomic inhibitory effect on basolateral IK(Ca) channels in crypt cells from normal human sigmoid colon. Patch clamp studies revealed that in cell-attached patches, IK(Ca) channel activity decreased progressively to 38+/-8% (P<0.001) of the basal value 10 min after the addition of 1 nmol/L aldosterone, and decreased further to 23+/-6% (P<0.02) of the basal value 5 min after increasing the aldosterone concentration to 10 nmol/L. Pre-incubation of crypts with 1 micromol/L chelerythrine chloride or 1 micromol/L Gö 6976 (PKC inhibitors) prevented the inhibitory effect of aldosterone. Conversely, channel activity decreased to 60+/-9% (P<0.02) of the basal value 10 min after the addition of 500 nmol/L PMA (a PKC activator), whereas 4alpha-PMA (an inactive ester) had no effect. When aldosterone (10 nmol/L) and PMA were added together, IK(Ca) channel activity was inhibited to the same extent as with aldosterone alone. These results indicate that aldosterone's non-genomic inhibitory effect on the macroscopic basolateral K(+) conductance in human colonic crypts reflects PKC-mediated inhibition of IK(Ca) channels.

TASK-2 is a member of the two-pore domain K+ (K-2P) channel family that is expressed at high levels in several epithelia, including the proximal tubule. In common with the other TASK channels, TASK-2 is sensitive to changes in extracellular pH. We have expressed human TASK-2 in Chinese hamster ovary cells and studied whole cell and single-channel activity by patch clamp. The open probability of K2P channels is generally independent of voltage, yielding linear current-voltage (I-V) curves. Despite these properties, we found that these channels showed distinct inward rectification immediately on the establishment of whole cell clamp, which became progressively less pronounced with time. This rectification was due to intracellular Na+ but was unaffected by polyamines or Mg2+ (agents that cause rectification in Kir channels). Rectification was concentration- and voltage-dependent and could be reversibly induced by switching between Na+-rich and Na+-free bath solutions. In excised inside-out patches, Na+ reduced the amplitude of single-channel currents, indicative of rapid block and unblock of the pore. Mutations in the selectivity filter abolished Na+-induced rectification, suggesting that Na+ binds within the selectivity filter in wild-type channels. This sensitivity to intracellular Na+ may be an additional potential regulatory mechanism of TASK-2 channels.